We propose to elucidate mechanisms of early development of the inner ear in the zebrafish, Danio rerio. The inner ear arises form a simple thickening on the surface of the embryo termed the otic placode, which subsequently forms the otic vesicle where sensory epithelia and neurons of the ear arise. We have shown that each of these processes requires Fgf signaling. At each step pax2/5/8 genes are induced by Fgf and help mediate or amplify Fgf signaling. In three specific aims we will examine how pax2/5/8 genes function and interact with other stage- and region-specific cofactors. 1) We will test specific functions of pax8 in otic placode induction and its interactions with coregulated genes sox3 and gbx2. 2) We will test a model in which Fgf stimulates ongoing formation of sensory epithelia by co-inducing pax2/5 genes, sox2 and proneural genes atoh1a and atoh1b (related to Math1). We hypothesize these genes regulate distinct aspects of specification, differentiation and survival of sensory hair cells. 3) We will test the role of Fgf and pax2/5 genes in regulating the expression and function of another proneural gene, neurog1, during formation of otic neuroblasts. Neuroblasts normally form in close proximity to sensory epithelia and we hypothesize that pax2/5 genes help distinguish the different responses to local Fgfs. To facilitate these aims, we will make use of heat shock- inducible transgenes to test the effects of misexpressing various genes at specific times and locations. Combined with loss-of-function approaches, these studies will provide fundamental insights into mechanisms of otic placode induction and sensory/neural development. These processes and regulatory functions are highly conserved. Moreover, studying the effects of misexpressing proneural genes in zebrafish could inform efforts to use similar approaches to restore hearing in mammals. PUBLIC HEALTH RELEVANCE Loss of hearing is a common affliction and results from permanent loss of sensory hair cells and/or neurons of the inner ear. It is widely believed that the key to restoring hearing in humans lies in reactivating the developmental processes by which hair cells and neurons first develop in the embryo. Using zebrafish as a model, we are investigating the functions of genes believed to regulate these processes in all vertebrates.